Intestinal Stem Cell Survival Research Articles

Nocardia rubra cell-wall skeleton mitigates whole abdominal irradiation-induced intestinal injury via regulating macrophage function.

Ionizing radiation (IR)-induced intestinal injury is a major side effect and dose-limiting toxicity in patients receiving radiotherapy. There is an urgent need to identify an effective and safe radioprotectant to reduce radiation-induced intestinal injury. Immunoregulation is considered an effective strategy against IR-induced injury. The purpose of this article was to investigate the protective effect of Nocardia rubra cell wall skeleton (Nr-CWS), an immunomodulator, on radiation-induced intestinal damage and to explore its potential mechanism. C57BL/6J male mice exposed to 12Gy whole abdominal irradiation (WAI) were examined for survival rate, morphology and function of the intestine and spleen, as well as the gut microbiota, to comprehensively evaluate the therapeutic effects of Nr-CWS on radiation-induced intestinal and splenetic injury. To further elucidate the underlying mechanisms of Nr-CWS-mediated intestinal protection, macrophages were depleted by clodronate liposomes to determine whether Nr-CWS-induced radioprotection is macrophage dependent, and the function of peritoneal macrophages stimulated by Nr-CWS was detected in vitro. Our data showed that Nr-CWS promoted the recovery of intestinal barrier function, enhanced leucine-rich repeat-containing G protein-coupled receptor 5+ intestinal stem cell survival and the regeneration of intestinal epithelial cells, maintained intestinal flora homeostasis, protected spleen morphology and function, and improved the outcome of mice exposed to 12Gy WAI. Mechanistic studies indicated that Nr-CWS recruited macrophages to reduce WAI-induced intestinal damage. Moreover, macrophage depletion by clodronate liposomes blocked Nr-CWS-induced radioprotection. In vitro, we found that Nr-CWS activated the nuclear factor kappa-B signaling pathway and promoted the phagocytosis and migration ability of peritoneal macrophages. Our study suggests the therapeutic effect of Nr-CWS on radiation-induced intestinal injury, and provides possible therapeutic strategy and potential preventive and therapeutic drugs to alleviate it.

PTBP1 controls intestinal epithelial regeneration through post-transcriptional regulation of gene expression.

The intestinal epithelial regeneration is driven by intestinal stem cells under homeostatic conditions. Differentiated intestinal epithelial cells, such as Paneth cells, are capable of acquiring multipotency and contributing to regeneration upon theloss of intestinal stem cells. Paneth cells also support intestinal stem cell survival and regeneration. We report here that depletion of an RNA-binding protein named polypyrimidine tract binding protein 1 (PTBP1) in mouse intestinal epithelial cells causes intestinal stem cell death and epithelial regeneration failure. Mechanistically, we show that PTBP1 inhibits neuronal-like splicing programs in intestinal crypt cells, which is critical for maintaining intestinal stem cell stemness. This function is achieved at least in part through promoting the non-productive splicing of its paralog PTBP2. Moreover, PTBP1 inhibits the expression of an AKT inhibitor PHLDA3 in Paneth cells and permits AKT activation, which presumably maintains Paneth cell plasticity and function in supporting intestinal stem cell niche. We show that PTBP1 directly binds to a CU-rich region in the 3' UTR of Phlda3, which we demonstrate to be critical for downregulating the mRNA and protein levels of Phlda3. Our results thus reveal the multifaceted in vivo regulation of intestinal epithelial regeneration by PTBP1 at the post-transcriptional level.

A DLG1-ARHGAP31-CDC42 axis is essential for the intestinal stem cell response to fluctuating niche Wnt signaling.

A central factor in the maintenance of tissue integrity is the response of stem cells to variations in the levels of niche signals. In the gut, intestinal stem cells (ISCs) depend on Wnt ligands for self-renewal and proliferation. Transient increases in Wnt signaling promote regeneration after injury or in inflammatory bowel diseases, whereas constitutive activation of this pathway leads to colorectal cancer. Here, we report that Discs large 1 (Dlg1), although dispensable for polarity and cellular turnover during intestinal homeostasis, is required for ISC survival in the context of increased Wnt signaling. RNA sequencing (RNA-seq) and genetic mouse models demonstrated that DLG1 regulates the cellular response to increased canonical Wnt ligands. This occurs via the transcriptional regulation of Arhgap31, a GTPase-activating protein that deactivates CDC42, an effector of the non-canonical Wnt pathway. These findings reveal a DLG1-ARHGAP31-CDC42 axis that is essential for the ISC response to increased niche Wnt signaling.

FUT2-dependent fucosylation of HYOU1 protects intestinal stem cells against inflammatory injury by regulating unfolded protein response

The intestinal epithelial repair after injury is coordinated by intestinal stem cells (ISCs). Fucosylation catalyzed by fucosyltransferase 2 (FUT2) of the intestinal epithelium is beneficial to mucosal healing but poorly defined is the influence on ISCs. The dextran sulfate sodium (DSS) and lipopolysaccharide (LPS) model were used to assess the role of FUT2 on ISCs after injury. The apoptosis, function, and stemness of ISCs were analyzed using intestinal organoids from WT and Fut2ΔISC (ISC-specific Fut2 knockout) mice incubated with LPS and fucose. N-glycoproteomics, UEA-1 chromatography, and site-directed mutagenesis were monitored to dissect the regulatory mechanism, identify the target fucosylated protein and the corresponding modification site. Fucose could alleviate intestinal epithelial damage via upregulating FUT2 and α-1,2-fucosylation of ISCs. Oxidative stress, mitochondrial dysfunction, and cell apoptosis were impeded by fucose. Meanwhile, fucose sustained the growth and proliferation capacity of intestinal organoids treated with LPS. Contrarily, FUT2 depletion in ISCs aggravated the epithelial damage and disrupted the growth and proliferation capacity of ISCs via escalating LPS-induced endoplasmic reticulum (ER) stress and initiating the IRE1/TRAF2/ASK1/JNK branch of unfolded protein response (UPR). Fucosylation of the chaperone protein HYOU1 at the N-glycosylation site of asparagine (Asn) 862 mediated by FUT2 was identified to facilitate ISCs survival and self-renewal, and improve ISCs resistance to ER stress and inflammatory injury. Our study highlights a fucosylation-dependent protective mechanism of ISCs against inflammation, which may provide a fascinating strategy for treating intestinal injury disorders.

The Wu-Shi-Cha formula protects against ulcerative colitis by orchestrating immunity and microbiota homeostasis

Ethnopharmacological relevanceUlcerative colitis (UC) has become a healthy burden worldwide due to its insidious onset and repetitive relapse, with a rather complex etiology, including inappropriate immune response, dysbiosis, genetic susceptibility, and unhealthy diets. The Wu-Shi-Cha (WSC) formula is a widely utilized drug to protect against gastrointestinal disorders. Aim of the studyThe study aspired to dissect the pertinent mechanisms of the WSC to treat UC. Materials and methodsNetwork pharmacology and weighted gene co-expression network analysis (WGCNA) were performed to predict the targets of WSC in the context of UC and colorectal cancer. Dextran sodium sulfate (DSS) was used to construct murine models of experimental colitis, and the WSC was given to colitis mice for 14 days. Feces and colon samples were subjected to 16S rRNA gene sequencing combined with liquid chromatography-mass spectrometry (LC-MS) and biochemical experiments, respectively. ResultsNetwork pharmacology analysis predicted that the WSC formula could orchestrate inflammation, infection, and tumorigenesis, and WGCNA based on The Cancer Genome Atlas (TCGA) database showed a potent anti-neoplastic effect of the WSC therapy for colorectal cancer. The WSC therapy rescued bursts of pro-inflammatory cytokines and colonic epithelial collapse in DSS-induced colitis mice. Moreover, the high dose of WSC treatment facilitated the alternative activation of peritoneal macrophages (Mφs) and these Mφs were conducive to the survival of intestinal stem cells (ISCs), and the disturbed homeostasis of gut microbiota was re-established after WSC treatment, as evidenced by the decreased colonization of pathological taxa in the fecal samples. ConclusionThe WSC formula suppresses inflammation and re-establishes the homeostasis of gut microbiota, thereby ameliorating colitis progression.

Autophagy in PDGFRα+ mesenchymal cells is essential for intestinal stem cell survival

Autophagy defects are a risk factor for inflammatory bowel diseases (IBDs) through unknown mechanisms. Whole-body conditional deletion of autophagy-related gene (Atg) Atg7 in adult mice (Atg7Δ/Δ) causes tissue damage and death within 3 mo due to neurodegeneration without substantial effect on intestine. In contrast, we report here that whole-body conditional deletion of other essential Atg genes Atg5 or Fip200/Atg17 in adult mice (Atg5Δ/Δ or Fip200Δ/Δ) caused death within 5 d due to rapid autophagy inhibition, elimination of ileum stem cells, and loss of barrier function. Atg5Δ/Δ mice lost PDGFRα+ mesenchymal cells (PMCs) and Wnt signaling essential for stem cell renewal, which were partially rescued by exogenous Wnt. Matrix-assisted laser desorption ionization coupled to mass spectrometry imaging (MALDI-MSI) of Atg5Δ/Δ ileum revealed depletion of aspartate and nucleotides, consistent with metabolic insufficiency underlying PMC loss. The difference in the autophagy gene knockout phenotypes is likely due to distinct kinetics of autophagy loss, as deletion of Atg5 more gradually extended lifespan phenocopying deletion of Atg7 or Atg12. Thus, autophagy is required for PMC metabolism and ileum stem cell and mammalian survival. Failure to maintain PMCs through autophagy may therefore contribute to IBD.

Disulfiram Protects Against Radiation-Induced Intestinal Injury in Mice.

Radiation-induced intestinal injury (RIII) occurs after high doses of radiation exposure. RIII restricts the therapeutic efficacy of radiotherapy in cancer and increases morbidity and mortality in nuclear disasters. Currently, there is no approved agent for the prevention or treatment of RIII. Here, we reported that the disulfiram, an FDA-approved alcohol deterrent, prolonged the survival in mice after lethal irradiation. Pretreatment with disulfiram inhibited proliferation within 24 h after irradiation, but improved crypt regeneration at 3.5 days post-irradiation. Mechanistically, disulfiram promoted Lgr5+ intestinal stem cells (ISCs) survival and maintained their ability to regenerate intestinal epithelium after radiation. Moreover, disulfiram suppresses DNA damage accumulation, thus inhibits aberrant mitosis after radiation. Unexpectedly, disulfiram treatment did not inhibit crypt cell apoptosis 4 h after radiation and the regeneration of crypts from PUMA-deficient mice after irradiation was also promoted by disulfiram. In conclusion, our findings demonstrate that disulfiram regulates the DNA damage response and survival of ISCs through affecting the cell cycle. Given its radioprotective efficacy and decades of application in humans, disulfiram is a promising candidate to prevent RIII in cancer therapy and nuclear accident.

Effects of Immune Cells on Intestinal Stem Cells: Prospects for Therapeutic Targets.

The intestinal epithelium undergoes rapid cell turnover to maintain the integrity of the mucosal barrier, which is driven by the proliferation and differentiation of intestinal stem cells (ISCs). Due to their properties, ISCs are not only vulnerable targets during intestinal damage, but also act as the resources responsible for repair and regeneration. Moreover, the intestinal tract is the largest immune organ in the body, with the greatest number of immune cells including, but not limited to, macrophages, innate lymphoid cells and T cells. With the advance of intestinal organoid culture systems and single-cell RNA sequencing, the effects of immune cells on ISCs have been initially explored. As a component of the stem cell niche, these activated immune cells and their corresponding cytokines directly modulate apoptosis or survival of ISCs, leading to either destruction or protection of the intestinal epithelium in immune-mediated diseases, such as inflammatory bowel disease and graft-versus-host disease. In this review, we describe the effects of various immune cells on ISCs, as well as the mechanisms underlying these effects. We also highlight the remarkable role of ISCs in intestinal pathogenesis and raise the possibility of developing novel and effective therapeutic strategies for immune-mediated diseases based on ISCs.

Aging of intestinal stem cells.

SummaryThe intestine is one of the organs that relies on stem cell function for maintaining tissue homeostasis. Recent findings on intestinal aging show that intestinal architecture, such as villus length, crypt size, and cell composition changes in the aged crypts. The correspondent decline in the regenerative capacity of the intestine is mainly due to a decline in intestinal stem cell function upon aging, as the underlying mechanisms of aging intestinal stem cells are beginning to unravel. This review summarizes our current knowledge on stem cell-intrinsic mechanisms of aging of intestinal stem cells and their connection to extrinsic factors, such as niche cells and microbiota and will introduce recent approaches to attenuate or even revert the aging of intestinal stem cells.

Cathelicidin-WA Protects Against LPS-Induced Gut Damage Through Enhancing Survival and Function of Intestinal Stem Cells.

Preservation of intestinal stem cells (ISCs) plays a critical role in initiating epithelial regeneration after intestinal injury. Cathelicidin peptides have been shown to participate in regulating intestinal damage repair. However, it is not known how exactly Cathelicidin-WA (CWA) exert its function after tissue damage. Using a gut injury model in mice involving Lipopolysaccharide (LPS), we observed that CWA administration significantly improved intestinal barrier function, preserved ISCs survival, and augmented ISCs viability within the small intestine (SI) under LPS treatment. In addition, CWA administration effectively prevented proliferation stops and promoted the growth of isolated crypts. Mechanistically, our results show that the appearance of γH2AX was accompanied by weakened expression of SETDB1, a gene that has been reported to safeguard genome stability. Notably, we found that CWA significantly rescued the decreased expression of SETDB1 and reduced DNA damage after LPS treatment. Taken together, CWA could protect against LPS-induced gut damage through enhancing ISCs survival and function. Our results suggest that CWA may become an effective therapeutic regulator to treat intestinal diseases and infections.

CBP/P300 Inhibitors Mitigate Radiation-Induced GI Syndrome by Promoting Intestinal Stem Cell-Mediated Crypt Regeneration.

Radiation-induced gastrointestinal syndrome (RIGS) is currently the main cause of death for people exposed to a high dose of irradiation during nuclear incidents, and there is currently no approved effective therapy. Here, we found that CBP/P300 inhibitors, with high efficacy and low toxicity, might be promising radiation mitigators that can cure RIGS. Exvivo 3D organoid cultures derived from mouse jejunum and human ileum and colon were used to examine the radio-mitigative effects of CBP/P300 inhibitors. The radio-mitigative effect was evaluated by quantifying the survival rate and size of organoids after radiation. SGC-CBP30 (50 mg/kg body weight), an inhibitor of CBP/P300, was intraperitoneally injected into C57B/6J mice 24 hours after subtotal-body irradiation or whole-body irradiation. The regenerated crypts and animal survival were determined by microcolony assay and the Kaplan-Meier method, respectively. Lgr5-lacZ mice were used to evaluate the survival of intestinal stem cells after treatments. We found that CBP/P300 inhibitors were effective mitigators that could be used to treat RIGS. CBP/P300 inhibition promoted the regeneration of intestinal organoids invitro and of crypts invivo. Remarkably, the administration of CBP/P300 inhibitors to mice 24 hours after lethal irradiation promoted Lgr5+ intestinal stem cell and crypt recovery, resulting in improved mouse survival. Moreover, our data show that CBP/P300 inhibitors rescued irradiated mice from RIGS by delaying intestinal epithelial cell cycle progression after radiation. These data demonstrate that CBP/P300 inhibitors are effective medical countermeasures to mitigate gastrointestinal toxicity from radiation.

Green Tea Derivative EGCG Protects Against Radiation Induced Intestinal Injury By Modulating Nrf2 Signaling Pathway

Intestine is one of the most radio-sensitive tissues, and radiation induced intestinal injury (RIII) remains one of the most prevalent dose-limiting toxicities in radiotherapy for pelvic and abdominal cancers. The failure of synthetic compounds for efficient prevention or treatment of RIII led to growing interest in the study of natural derivatives from nontoxic plants. Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, is a free radical scavenger and potent antioxidant agent, and has been widely studied in cancer chemoprevention. However, whether EGCG has the therapeutic effect on RIII remains unknown. Male C57BL/6J mice were treated with 9Gy of total body irradiation (TBI). 25mg/kg of EGCG were intraperitoneally injected before (once a day for 5 days) and 30minutes after TBI. Then, the survival rate and body weight were recorded. Meanwhile, jejunum tissues were collected and the severity of RIII was measured by HE staining, immunohistochemistry (IHC), immunofluorescence (IF) and TUNEL. Next, human intestinal epithelial cells (HIEC) were treated with EGCG and irradiated. Then, colony formation assay, flow cytometry, IF were conducted, and the level of reactive oxygen species (ROS) was assessed. Further, the expression of pivotal anti-oxidative response regulator nuclear factor erythroid 2 related factor 2 (Nrf2) was detected by IF and western blotting. Compared to the control, EGCG treatment significantly improved the survival time (from 6.1days to 10.4days, P<0.05) and increased the body weights of mice after TBI. HE staining of jejunum tissues showed that EGCG mitigated RIII, as reflected by the dramatic attenuation of crypt-villi architecture destruction. IHC results showed that EGCG treatment remarkably increased the Ki67-positive (from 15.7 to 30.1, P<0.001) and Lgr5-positive (from1.5 to 3.0, P<0.001) cells per crypt, indicating that EGCG promoted proliferation and survival of intestinal stem cells after TBI. Meanwhile, in EGCG treatment group the number of TUNEL-positive cells (from 4.9 to 1.0, P<0.001), γH2AX foci (from 2.8 to 0.4, P<0.001) and 8-OHdG-positive cells (from 3.5 to 0.6, P<0.001) significantly decreased per crypt, indicating that EGCG inhibited radiation-induced apoptosis and DNA damage in intestine. Consistently, the EGCG treatment remarkably improved the colony forming ability, while decreased apoptosis ratio, γH2AX foci and 8-OHdG foci of irradiated HIEC cells. For the mechanism study, EGCG treatment diminished the radiation-induced production of ROS, and promoted the nuclear translocation of key anti-oxidative response regulator Nrf2 of irradiated HIEC cells, indicating that EGCG scavenged radiation-induced ROS, and subsequently attenuated ROS-regulated intestinal oxidative DNA damage. Collectively, our findings suggested that EGCG protects against RIII by reducing ROS-induced DNA damage, and might be a novel therapeutic option for the clinical treatment of RIII.

Abstract 3745: Mechanisms of HIF2-mediated small intestine radioprotection

Abstract Pancreatic cancer is the fourth-leading cause of cancer-related deaths in the United States, and it is projected to become the second by 2030. The 5-year overall survival rate for locally advanced pancreatic cancer (LAPC) remains &amp;lt; 5%. Chemotherapy regimens often fail due to poor drug delivery and penetration into the tumor. Because surgery is not feasible by definition in LAPC, radiation therapy (RT) is the only option to achieve local control. However, irradiating the pancreas is challenging because potentially curative doses are limited by the neighboring small intestine, which can only tolerate a maximum of 50Gy. A selective radioprotector of the intestinal tract could allow pancreatic cancer patients to receive a higher, and potentially definitive, dose of radiation by increasing the duodenum’s tolerance to survive radiation injury. The EGLN prolyl hydroxylases are cellular oxygen sensors that regulate cell survival and metabolism through the degradation of hypoxia-inducible factors (HIFs) in the presence of oxygen. Our group has shown that HIF2 stabilization through genetic or pharmacologic inhibition of the EGLN family prevents death from radiation-induced gastrointestinal toxicity in mice without protecting the primary tumor. To understand the mechanism by which HIF2 mediates this protection, we generated intestinal organoids from mice with a Cre-inducible allele that expresses a non-degradable form of human HIF1 or HIF2. Whole transcriptomic analysis revealed HIF1 and HIF2 have distinct gene responses in the small intestinal crypt. HIF2 induced the expression of known radiation modulators and genes involved in oxidative damage response, tissue healing, and intestinal homeostasis such as the non-canonical Wnt5a. We validated HIF2 drives Wnt5a expression in multiple organoid model systems. We then generated knockouts (KO) of Wnt5a or Ror2, its cognate receptor, using the Cre/lox system to test if Wnt5a is necessary and sufficient for radioprotection. By using the spheroid formation assay, a modified clonogenic assay, we found Wnt5a KO organoids had decreased crypt regeneration following RT. Our preliminary data suggest Wnt5a protects the duodenum from RT by increasing intestinal stem cell survival. Citation Format: Carolina J. Garcia Garcia, Suman Govindaraju, Marimar de la Cruz Bonilla, Cullen M. Taniguchi. Mechanisms of HIF2-mediated small intestine radioprotection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3745.

Combination treatment of podophyllotoxin and rutin promotes mouse Lgr5+ ve intestinal stem cells survival against lethal radiation injury through Wnt signaling.

It has been well established that radiation-induced gastrointestinal injury is manifested through loss of intestinal crypt stem cells and disruption of the mucosal layers, resulting in diarrhoea, weight loss, electrolyte imbalance, infection and mortality. Podophyllotoxin and rutin in combination (G-003M) has been reported to regulate endogenous cellular antioxidant defense systems and inflammatory response. However, the mechanism by which G-003M ameliorates radiation-induced intestinal stem cell (ISC) injury remains unclear. Here, we hypothesize the radioprotective potential of G-003M would amplify the intestinal crypt stem cells through upregulation of Wnt/β-catenin signaling and accelerate the reconstitution of the irradiated intestine. Our results showed significant functional and structural intestine regeneration in irradiated animals following G-003M treatment which resulted in improved animal survival. Immunohistochemical examination revealed an enhancement in Lgr5+ ve crypt stem cells. Increased β-catenin nuclear translocation resulted in upregulation of β-catenin target genes that supported ISC renewal and expansion in G-003M-treated mice, as compared to IR-treated mice. However, G-003M could not rescue the Wnt knockdown cohorts (XAV939 treated) which exhibited greater incidence of intestinal apoptosis, DNA damage and crypt depopulation upon radiation exposure. These findings suggest the involvement of Wnt pathway during G-003M mediated amelioration of IR-induced ISC injury. G-003M also minimised acute inflammation by restricting the infiltration of immune cells into the intestinal venules. Furthermore, G-003M treated animals showed improved anti-tumor response compared to FDA approved Amifostine. Taken together, our findings suggest that G-003M may be used as a potential countermeasure for radiation injuries as well as an adjuvant during anti-cancer therapy.

PERK is essential for proliferation of intestinal stem cells in mice

Protein kinase RNA-like Endoplasmic Reticulum Kinase (PERK) is an endoplasmic reticulum stress sensor that possesses pro-survival capability and contributes to cell homeostasis and survival. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) has been recognized as a stem cell marker in intestinal epithelial cells. To determine whether PERK modulates the proliferation of intestinal stem cells, we investigated the effects of PERK knock-down on intestinal Lgr5-positive stem cells in mice. Lgr5-EGFP knock-in mice were fed with lentivirus-PERK shRNA twice a day for three days. Isolated intestinal Lgr5-positive stem cells were treated with lentivirus-PERK shRNA. The number of Lgr5-positive cells, the proliferation and apoptotic indices, several biomarkers for proliferation and differentiation, and Akt expression in intestinal stem cells were detected in vivo, in vitro and in two intestinal epithelial injury models caused by radiotherapy and sepsis. PERK knock-down could significantly diminish the number and proliferation of Lgr5-positive cells, induce the low expression of several proliferation markers and the high expression of several differentiation markers in Lgr5-positive cells, enhance the apoptotic Lgr5-positive cells, and reduce the Akt expression in intestinal Lgr5-positive stem cells. Similar results were observed in radiotherapy- and sepsis-induced intestinal injuries. Moreover, PERK inhibition markedly decreased the survival of mice in response to radiation and sepsis. These results suggest a critical role for PERK in the proliferation and survival of intestinal stem cells in mice.

Abstract 4164: Fasting protects mice from lethal radiation by promoting small intestinal stem cell survival

Abstract Pancreatic cancer is the fourth leading cause of cancer-related deaths in the US. Surgical resection is the only potentially curative treatment; however, only 15%-20% of patients present with tumors that can be resected. There is no consensus regarding standard of care in unresectable cases, however many academic centers use stereotactic body radiotherapy (SBRT) to give tumor-directed radiotherapy (RT). Unfortunately, even this conformal technique can still cause severe gastrointestinal (GI) toxic effects caused by the proximity of the pancreatic head to the duodenum. Protecting the intestine from the toxic effects of radiation may enable dose escalation that could achieve more effective local control of disease. We and others have previously shown that a prolonged fast of 24 hours protects mice from lethal doses of etoposide. In this study, we extend and build on our previous finding to demonstrate that a similar 24 hour fast also protects from lethal doses of total abdominal radiation. Histologic analyses, using the Withers-Elkind microcolony assay, show that fasting protected small intestinal (SI) stem cells from radiation damage and promoted early regeneration. To show a proof-of-principle for the use of this radioporotective maneuver in cancer therapy, we developed an orthotopic model of pancreatic cancer using KPC tumor cells syngeneic to C57BL/6. Here, we show that fasting-mediated intestinal protection enabled dose escalated SBRT for treatment of these orthotopic tumors. RT with fasting radioprotection delayed tumor growth and improved survival compared to controls. Given this robust phenotype, we developed a 3D culture ex vivo assay using intestinal stem cell enriched epithelial spheroid cultures. We modified these intestinal spheroids with a bioluminescent reporter and used these cells to develop a modified clonogenic assay for 3D culture that can be used to identify novel radioprotectors, such as a fasting mimetic. Taken together, these results suggest that fasting protects small intestinal stem cells sufficiently to allow animals to receive potentially curative doses of abdominal radiation that would other wise be lethal. Future work will aim to identifying the mechanism by which fasting confers intestinal protection and drug candidates that can be used to mimic this fasting-mediated protection. Citation Format: Marimar de la Cruz Bonilla, Kristina M. Stemler, Tara N. Fujimoto, Sabrina Jeter-Jones, Jessica M. Molkentine, Gabriela M. Asencio Torres, Cullen M. Taniguchi, Helen Piwnica-Worms. Fasting protects mice from lethal radiation by promoting small intestinal stem cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4164.

The histone variant macroH2A confers functional robustness to the intestinal stem cell compartment.

A stem cell’s epigenome directs cell fate during development, homeostasis, and regeneration. Epigenetic dysregulation can lead to inappropriate cell fate decisions, aberrant cell function, and even cancer. The histone variant macroH2A has been shown to influence gene expression, guide cell fate, and safeguard against genotoxic stress. Interestingly, mice lacking functional macroH2A histones (hereafter referred to as macroH2A DKO) are viable and fertile; yet suffer from increased perinatal death and reduced weight and size compared to wildtype (WT). Here, we ask whether the ostensible reduced vigor of macroH2A DKO mice extends to intestinal stem cell (ISC) function during homeostasis, regeneration, and oncogenesis. Lgr5-eGFP-IRES-CreERT2 or Hopx-CreERT2::Rosa26-LSL-tdTomato ISC reporter mice or the C57BL/6J-Apcmin/J murine intestinal adenoma model were bred into a macroH2A DKO or strain-matched WT background and assessed for ISC functionality, regeneration and tumorigenesis. High-dose (12Gy) whole-body γ-irradiation was used as an injury model. We show that macroH2A is dispensable for intestinal homeostasis and macroH2A DKO mice have similar numbers of active crypt-base columnar ISCs (CBCs). MacroH2A DKO intestine exhibits impaired regeneration following injury, despite having significantly more putative reserve ISCs. DKO reserve ISCs disproportionately undergo apoptosis compared to WT after DNA damage infliction. Interestingly, a macroH2A DKO background does not significantly increase tumorigenesis in the Apcmin model of intestinal adenoma. We conclude that macroH2A influences reserve ISC number and function during homeostasis and regeneration. These data suggest macroH2A enhances reserve ISC survival after DNA damage and thus confers functional robustness to the intestinal epithelium.

Covering the Cover

Covering the Cover

Abstract 918: Cdc42 is crucial for intestinal stem cells survival by regulating Wnt and YAP signaling

Abstract Strictly defined architecture of intestinal epithelial cells and the gradient of signaling cues along the villus-crypt axis ensure the maintenance of adult intestinal stem cells, as well as proper regeneration and differentiation of all intestinal epithelial cell types. The Rho family small GTPase Cdc42 controls the polarized division of intestinal stem cells, and affects subsequent fate-determination of the progenitor cells. As we previously described in Cdc42 Villin-Cre knockout mice, loss of Paneth cells from crypt bottom and emergence of Goblet cell-like intermediate cells at the villus indicate possible compromise of the canonical Wnt signaling during Paneth cell differentiation. Here we further characterized the underlying molecular mechanism of Cdc42 regulating intestinal stem cell survival, proliferation, and differentiation. IHC of the intestinal tissue in Cdc42 Villin-Cre knockout mice suggests greatly reduced Olfm4 level in the stem cells, which are abnormally positioned in the crypts, along with a reduction of both transcription co-factors beta-Catenin and YAP. However, the Notch signaling, which is required for stem cells and the differentiation of absorptive linage, seems intact. The autonomous regulation of Cdc42 on Yap signaling is probably through its specific role in stabilizing cell-cell junction and actin filaments in the epithelium, both of which are known regulatory inputs of the Hippo-YAP pathway. Both Wnt/beta-Catenin and Yap signaling are required for the initiation and progression of colorectal cancer, and the regeneration of stem cells post radiation. Given that Cdc42 is overexpressed in multiple types of cancers, especially colorectal cancer, our finding that Cdc42 regulates both Wnt and YAP pathways in the homeostasis of adult intestinal tissue sheds new light on the molecular mechanism of its pathological involvement, and potential therapeutic target. Citation Format: Xiao Zhang, Nan Gao. Cdc42 is crucial for intestinal stem cells survival by regulating Wnt and YAP signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 918. doi:10.1158/1538-7445.AM2017-918

Probiotic Lactobacillus acidophilus bacteria or synthetic TLR2 agonist boost the growth of chicken embryo intestinal organoids in cultures comprising epithelial cells and myofibroblasts.

The intestinal epithelial cells reside in close proximity to myofibroblasts and microbiota, which are supposed to have an impact on intestinal stem cells fate and to influence processes of tissue maturation and regeneration. Mechanism underlying these phenomena and their diversity among vertebrates can be studied in 3D organoid cultures. We investigated the growth of chicken embryo intestinal epithelial organoids in Matrigel with and without Toll-like receptors (TLRs) stimulation. The organoid cultures contained also some myofibroblasts with potential to promote intestinal stem cell survival. Organoid cells, expressing TLR4, TLR2 type 1 and TLR2 type 2 were incubated with their agonists (lipopolysaccharide - LPS and Pam3CSK4) or co-cultured with Lactobacillus acidophilus bacteria (LA-5). Pam3CSK4 and LA-5 promoted organoid growth, which was demonstrated by comparing the morphological parameters (mean number and area of organoids). The profile of prostaglandins (PG), known to promote intestinal regeneration, in supernatants from organoid and fibroblast cultures were evaluated. Both PGE2 and PGD2 were detected. As compared to unstimulated controls, supernatants from the Pam3CSK4-stimulated organoids contained twice as much of PGE2 and PGD2. The changes in production of prostaglandins and the support of epithelial cell growth by myofibroblasts are factors potentially responsible for stimulatory effect of TLR2 activation.